Chugai Seiyaku Kabushiki Kaisha

Yuichi Hirata

Masayuki Tsuchiya

Koh Sato

Hiroyuki Tsunoda

Kiyotaka Nakano

Junichi Nezu

Tetsuro Orita

Shinichi Hashimoto

Kouji Matsushima

Kenji Yoshida

Kiyoshi Habu

Kazuyuki Ojima

Purified immunocytes were analyzed for expression frequencies, and the NKIR gene expressed specifically in natural killer (NK) cells was successfully identified. The NKIR gene encodes a receptor. Agonists and antagonists for the receptor can be identified by using the receptor.

Proteins expressed in NK cells

Anti-human Mpl antibodies were isolated and purified. Anti-human Mpl diabodies and anti-human Mpl sc(Fv)2 were prepared using genetic engineering techniques and anti-human Mpl sc(Fv)2 was also humanized. The diabodies and sc(Fv)2 were assayed for TPO-like agonistic activity, and were found to have activities higher than those of anti-human Mpl antibodies, or activities equivalent to or higher than those of naturally-occurring human TPO ligand.

Anti-Mpl antibodies

Anti-MPL antibodies

A targeting vector was constructed by replacing exon regions in the SGRF gene with appropriate drug marker genes. This vector was transfected into mouse ES cell lines to obtain chimeric mice, which were then crossed with C57BL/6J mice to obtain mice comprising cells in which one SGRF gene alleles was inactivated. By crossing these mice with each other, the present inventors succeeded in producing mice in which both SGRF gene alleles were inactivated. These genetically modified animals can be used to predict the side effects of drugs such as SGRF antagonists.

SGRF gene-modified mouse

The invention relates to VEGF-like nucleic acid sequences, their use, and vectors and transformants containing such sequences.

Nucleic acid sequences encoding VEGF-like factors and uses thereof

A full-length cDNA corresponding to an EST (AA418955), which does not show any homology to other proteins in the database but has a weak homology to G-CSF, has been successfully isolated by synthesizing primers based on the EST sequence, and effecting PCR-cloning from a human fetal spleen library. Sequencing of the thus-isolated cDNA and analysis of its structure revealed that the cDNA has typical characteristics of a factor belonging to the IL-6/G-CSF/MGF family. It is also found out that the culture supernatant of said sequence-transfected CHO cells shows a proliferation supporting activity towards bone marrow cells in the coexistence of kit ligand.

Antibodies that bind to a member of the IL-6/G-CSF/MGF family

The present inventors used antibody engineering techniques to prepare functional antibodies that correspond to individual mutations in causative genes of diseases, and discovered that such antibodies enable the treatment of the diseases. Specifically, the inventors succeeded in preparing ligands, particularly minibodies, which have agonistic activity to receptors that have almost completely lost responsiveness to their natural ligands because of gene mutations (for example, a thrombopoietin (TPO) receptor whose reactivity to TPO has been markedly impaired), and which can transduce signals by interacting with these mutant receptors at levels comparable to normal.

Ligand having agonistic activity to mutated receptor

Several ESTs deduced as a part of a cDNA encoding a protein that is homologous with a membrane-bound steroid binding protein PMBP were found. Based on the sequence data of these ESTs, a consensus sequence was extracted, and primers were designed based on this consensus sequence. Using the thus designed primers, a polymerase chain reaction of human genes was effected. As a result, a gene encoding a novel steroid hormone binding protein that is homologous with PMBP was successfully isolated from a human for the first time.

Steroid hormone binding protein

A full-length cDNA corresponding to an EST (AA418955), which does not show any homology to other proteins in the database but has a weak homology to G-CSF, has been successfully isolated by synthesizing printers based on the EST sequence, and effecting PCR-cloning from a human fetal spleen library. Sequencing of the thus-isolated cDNA and analysis of its structure revealed that the cDNA has typical characteristics of a factor belonging to the IL-6/G-CSF/MGF family. It is also found out that the culture supernatant of said sequence-transfected CHO cells shows a proliferation supporting activity towards bone marrow cells in the coexistence of kit ligand.

Nucleic acids encoding cytokine-like proteins that promote cell proliferation

A novel human gene having a significant homology with a VEGF-C gene, a member of the VEGF family, has been isolated by the PCR method using primers designed based on the sequence of EST that is assumed to be homologous with the C-terminal region of the VEGF-C gene. Mouse and rat genes have been isolated based on the human gene isolated as above. A protein encoded by the above human gene has been isolated by introducing the gene into and expressing it. The isolated protein and genes can be applied to, for example, gene therapy for the VEGF-D deficiency, wound healing, and promotion of collateral vessel formation. Furthermore, VEGF-D protein inhibitors can be used as a novel anticancer drug, etc.

Growing community of inventors

Ibaraki, Japan

Yuichi Hirata